Elucidation of the mechanisms which regulate the concentrations of intracellular proteins in animal cells will add considerably to our understanding of normal cell function as well as to our understanding of the breakdown of regulatory processes which occurs in certain diseases, including cancer. We propose to use the isoenzymes of aldolase as a model system to investigate these regulatory mechanisms. The three aldolase subunit types, A, B, and C, are structurally homologous and were presumably derived from the same ancestral molecule; yet, the intracellular concentrations of the subunit types are independently regulated. I have previously shown that regulation at the level of subunit synthesis is primarily involved in determing the tissue-specific and stage-specific isoenzyme patterns of animal cells. We now want to investigate the regulation of subunit synthesis in detail. We will synthesize the three aldolases in cell-free protein synthesizing systems using mRNA derived from different tissues of adult and developing animals. We hope to correlate the levels of translatable subunit mRNAs with the rates of subunit synthesis observed in vivo. Further, we plan to investigate the possibility of translational regulation of subunit synthesis by sizing aldolase synthesizing polysomes and by testing the effects of initiation factors (or other components) derived from different tissues on the rates of translation of subunit mRNAs. Finally, we will further investigate the mechanism of aldolase subunit association by translating mRNAs of different tissues in the same cell-free incubation mixtures. The methods developed here can be directly applied to investigations on the altered regulation of isoenzyme concentrations which is associated with cancer and other disease states.